Functionally distinct populations of murine intestinal stem cells (ISCs), including Lgr5- and telomerase (mTert)-expressing cells, maintain the highly self-renewing intestinal epithelium. Lgr5+ cells are rapidly cycling and play an important role in daily homeostasis, whereas mTert+ cells are slowly cycling, highly resistant to injury, and play a role in intestinal regeneration. While the discovery of distinct populations of intestinal stem cells represents an important advance in our understanding of intestinal homeostasis, little is known regarding how these cells respond to the physiologic stress that results from fasting and refeeding. Regulated by complex and still poorly understood mechanisms, fasting has significant and rapid effects on the intestines. Increased epithelial cell apoptosis and decreased crypt cell proliferation lead to intestinal mucosal atrophy, which rapidly resolves upon re-feeding. Based on this, we hypothesized that during fasting, all ISC populations would be down regulated, however, our preliminary data revealed a dramatic induction of mTert+ ISCs. This led to the hypothesis that slowly cycling mTert+ ISCs play an important role in readying the small intestine for intestinal regeneration following fasting. Additionally, we hypothesize that Lgr5-expressing ISCs will be suppressed by fasting but will play a complementary role during the regenerative response. Studies proposed in Aim 1 and 2 are designed to characterize the role of slowly cycling mTert+ and rapidly cycling Lgr5+ ISCs, respectively, in response to the physiologic stress of fasting and re-feeding. In addition, these studies will employ gene expression profiling to investigate the underlying genetic mechanisms regulating their behavior. In summary, this proposal seeks to characterize the role of mTert+ and Lgr5+ ISCs in response to intestinal stress as modeled by fasting and to define the underlying regulatory mechanisms at the transcriptional level. A more comprehensive understanding of the mechanisms regulating the response of ISCs to stressful stimuli has potentially important implications for the prevention and treatment of multiple medical conditions including inflammatory bowel disease, as well as chemotherapy- and radiation-induced mucositis, common causes of morbidity amongst pediatric and adult patients. Although this application is narrowly focused on the role of ISCs during fasting, these studies may ultimately give rise to improved therapeutic treatment options for genetic, inflammatory, or post-surgical gastrointestinal conditions. In conjunction with a well-designed training program and highly supportive mentors, this application will help provide the foundation for a rigorous scientific career. PUBLIC HEALTH RELEVANCE: The goal of this project is to better understand how slowly- and rapidly-cycling intestinal stem cells protect the intestine against stress. Characterization of these cells will provide unique insight into the mechanisms regulating intestinal growth, regeneration and adaptation to the rapidly changing and stressful gut environment relevant to intestinal disease. Analysis of gene expression will identify key regulators of stem cell function and provide a basis to determine the critical changes leading to intestinal regeneration.